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盐藻小G蛋白DsRab的原核表达及纯化 被引量:1
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作者 李秀娟 柴晓杰 +2 位作者 陶晓迎 赵欢 丛玉婷 《生物技术通报》 CAS CSCD 北大核心 2014年第4期176-180,共5页
前期对盐藻小G蛋白基因DsRab研究表明,在盐胁迫诱导下该基因转录水平明显提高。为进一步研究该蛋白在盐藻耐盐机制中的作用,PCR扩增DsRab的开放阅读框(ORF),并将其克隆至带有GST标签的原核表达载体pGS-21a,得到重组表达载体pGS-21a-DsRa... 前期对盐藻小G蛋白基因DsRab研究表明,在盐胁迫诱导下该基因转录水平明显提高。为进一步研究该蛋白在盐藻耐盐机制中的作用,PCR扩增DsRab的开放阅读框(ORF),并将其克隆至带有GST标签的原核表达载体pGS-21a,得到重组表达载体pGS-21a-DsRab。将重组表达载体转化E.coli BL21(DE3),IPTG诱导表达,并优化诱导表达条件,利用GST-SefinoseTM Kit进行纯化,用SDS-PAGE和Western blot鉴定。结果表明,成功构建了重组表达载体pGS-21a-DsRab,SDS-PAGE结果显示得到的蛋白与预期分子量相符,并且纯度较高;Western blot检测结果初步证明该融合蛋白为GST-DsRab。 展开更多
关键词 盐藻 dsrab GST 原核表达 纯化
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A hot origin of dissimilatory sulfite reduction catalyzed by DsrAB in the Paleoarchean Era
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作者 Lingyun Tang Zhenhao Luo +8 位作者 Shaoming Gao Zhiliang Lin Mengqi Sun Runsheng Li Shu-Hong Gao Geng Wu Yiliang Li Linan Huang Lu Fan 《mLife》 2026年第1期108-121,共14页
Dissimilatory sulfite reduction(DSR)has been essential to microbial energy metabolism in the biogeochemical sulfur cycle since the Paleoarchean Era.However,due to the lack of an integrated assessment of geological rec... Dissimilatory sulfite reduction(DSR)has been essential to microbial energy metabolism in the biogeochemical sulfur cycle since the Paleoarchean Era.However,due to the lack of an integrated assessment of geological record and genomic data,the evolutionary origin of DSR remains elusive in terms of time,habitat,and genetic basis.In this study,we reconstructed the evolutionary pathways and the ancestral sequences of Dsr proteins by mining metagenomes ranging from mesothermal to hyperthermal environments.A phylogenetic analysis of the key catalytic enzyme,DsrAB,and other Dsr proteins indicates that the earliest and most basic functional cascade,DsrABCNM,emerged prior to the latest common ancestor of several basal branching DsrAB clusters encoded by bacteria and archaea.Using a molecular dating strategy that calibrates the protein tree with a species tree,we predicted that the DSR originated 3.508 billion years ago(Ga).This finding strongly confirms the earliest geological evidence of DSR(~3.47 Ga).Further predictions from ancestral sequence reconstruction indicate that the optimal catalytic temperature of DsrA at the time of DSR origin was approximately 73℃,which is consistent with the petrographic and geochemical evidence in early Archean hydrothermal deposits.After its hot origin,DsrA diversified into subclades that adapted to various temperature levels following the Great Oxidation Event.This is exemplified by the evolution of the reductive archaeal-type DsrA.Our results synchronize the molecular ages with the geological record,which advances our understanding of the earliest DSR systems and highlights the enzymatic adaptations of microbial life in the Archean biosphere. 展开更多
关键词 archaeal evolution dsrab METAGENOMICS sulfite reduction sulfur cycling
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